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SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells
Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. However, it remains technically challenging to analyze both the genome and transcriptome in the same ce...
Autores principales: | , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749184/ https://www.ncbi.nlm.nih.gov/pubmed/29208629 http://dx.doi.org/10.1101/gr.223263.117 |
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author | Han, Kyung Yeon Kim, Kyu-Tae Joung, Je-Gun Son, Dae-Soon Kim, Yeon Jeong Jo, Areum Jeon, Hyo-Jeong Moon, Hui-Sung Yoo, Chang Eun Chung, Woosung Eum, Hye Hyeon Kim, Sangmin Kim, Hong Kwan Lee, Jeong Eon Ahn, Myung-Ju Lee, Hae-Ock Park, Donghyun Park, Woong-Yang |
author_facet | Han, Kyung Yeon Kim, Kyu-Tae Joung, Je-Gun Son, Dae-Soon Kim, Yeon Jeong Jo, Areum Jeon, Hyo-Jeong Moon, Hui-Sung Yoo, Chang Eun Chung, Woosung Eum, Hye Hyeon Kim, Sangmin Kim, Hong Kwan Lee, Jeong Eon Ahn, Myung-Ju Lee, Hae-Ock Park, Donghyun Park, Woong-Yang |
author_sort | Han, Kyung Yeon |
collection | PubMed |
description | Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. However, it remains technically challenging to analyze both the genome and transcriptome in the same cell. Here, we report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations, such as copy-number and single-nucleotide variations, were more accurately captured by single-cell SIDR-seq compared with conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level. |
format | Online Article Text |
id | pubmed-5749184 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-57491842018-01-19 SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells Han, Kyung Yeon Kim, Kyu-Tae Joung, Je-Gun Son, Dae-Soon Kim, Yeon Jeong Jo, Areum Jeon, Hyo-Jeong Moon, Hui-Sung Yoo, Chang Eun Chung, Woosung Eum, Hye Hyeon Kim, Sangmin Kim, Hong Kwan Lee, Jeong Eon Ahn, Myung-Ju Lee, Hae-Ock Park, Donghyun Park, Woong-Yang Genome Res Method Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. However, it remains technically challenging to analyze both the genome and transcriptome in the same cell. Here, we report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations, such as copy-number and single-nucleotide variations, were more accurately captured by single-cell SIDR-seq compared with conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level. Cold Spring Harbor Laboratory Press 2018-01 /pmc/articles/PMC5749184/ /pubmed/29208629 http://dx.doi.org/10.1101/gr.223263.117 Text en © 2018 Han et al.; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by/4.0/ This article, published in Genome Research, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Method Han, Kyung Yeon Kim, Kyu-Tae Joung, Je-Gun Son, Dae-Soon Kim, Yeon Jeong Jo, Areum Jeon, Hyo-Jeong Moon, Hui-Sung Yoo, Chang Eun Chung, Woosung Eum, Hye Hyeon Kim, Sangmin Kim, Hong Kwan Lee, Jeong Eon Ahn, Myung-Ju Lee, Hae-Ock Park, Donghyun Park, Woong-Yang SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells |
title | SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells |
title_full | SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells |
title_fullStr | SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells |
title_full_unstemmed | SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells |
title_short | SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells |
title_sort | sidr: simultaneous isolation and parallel sequencing of genomic dna and total rna from single cells |
topic | Method |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749184/ https://www.ncbi.nlm.nih.gov/pubmed/29208629 http://dx.doi.org/10.1101/gr.223263.117 |
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